Genetic and pharmacological reactivation of the mammalian inactive X chromosome

Abstract

X-chromosome inactivation (XCI), the random transcriptional silencing of one X chromosome in somatic cells of female mammals, is a mechanism that ensures equal expression of X-linked genes in both sexes. XCI is initiated in cis by the noncoding Xist RNA, which coats the inactive X chromosome (Xi) from which it is produced. However, trans-acting factors that mediate XCI remain largely unknown. Here, we perform a large-scale RNA interference screen to identify trans-acting XCI factors (XCIFs) that comprise regulators of cell signaling and transcription, including the DNA methyltransferase, DNMT1. The expression pattern of the XCIFs explains the selective onset of XCI following differentiation. The XCIFs function, at least in part, by promoting expression and/or localization of Xist to the Xi. Surprisingly, we find that DNMT1, which is generally a transcriptional repressor, is an activator of Xist transcription. Small-molecule inhibitors of two of the XCIFs can reversibly reactivate the Xi, which has implications for treatment of Rett syndrome and other dominant X-linked diseases. A homozygous mouse knockout of one of the XCIFs, stanniocalcin 1 (STC1), has an expected XCI defect but surprisingly is phenotypically normal. Remarkably, X-linked genes are not overexpressed in female Stc1 −/− mice, revealing the existence of a mechanism(s) that can compensate for a persistent XCI deficiency to regulate X-linked gene expression.-chromosome inactivation (XCI), the random transcriptional silencing of one X chromosome in somatic cells of female mammals, is a mechanism that ensures equal expression of X-linked genes in both sexes (1). XCI is initiated by X inactive specific transcript (Xist), a 17-kb noncoding RNA whose expression during early embryogenesis is both necessary and sufficient for silencing (2, 3). Xist represses transcription in cis by coating only the X chromosome from which it is produced. Once Xist has been up-regulated during early development or differentiation, it continues to be expressed from the inactive X (Xi) even in fully differentiated somatic cells. Before the initiation of XCI, TSIX transcript, XIST antisense RNA (Tsix) an antisense repressor of Xist, blocks Xist up-regulation on the future active X chromosome (Xa) (4).An understanding of the factors and mechanisms involved in XCI is directly relevant to certain human diseases. In particular, loss-of-function mutations in the X-linked methyl-CpG binding protein 2 (MECP2) gene lead to the neurodevelopmental disorder Rett syndrome (RTT) (5-7). Most RTT patients are females who are heterozygous for MECP2 deficiency due to random XCI. Significantly, in a mouse model of RTT, reactivation of wild-type Mecp2 expression can reverse the disease phenotype even in late-stage adult animals (8). Thus, reactivation of the silenced wild-type MECP2 allele is a potential strategy for treating RTT.We have previously demonstrated how large-scale short hairpin RNA (shRNA) screens can be used to identify factors involved in epigenetic silencing of tumor suppressor ge...

Screen for reactivation of MeCP2 on the inactive X chromosome identifies the BMP/TGF-β superfamily as a regulator of XIST expression

“…Other kinases have also been identified in the screens: (i) PI3-kinase signaling network, including receptorassociated tyrosine kinase (Erb4), proximal kinases (e.g., PI3K and PDPK1), and a distal member (SGK2); (ii) protein kinase C member PRKD3; and (iii) mitotic kinases PLK2 and Aurka. Consistent with the idea that pharmacologic inhibition of kinase signaling networks identified in our screen can reactivate the Xi, work by other groups has demonstrated reactivation of Xi genes by smallmolecule inhibitors of PDPK1 and PI3K (22). Furthermore, Aurora kinase inhibitors have been identified in small-molecule screens for Xi reactivation (22,46).…”

“…3A). Whereas knockdown of several members of the PI3K/AKT group exhibited modestly reduced XIST levels (Dataset S2), as was previously observed for PDPK1 (22), the magnitude of the effect was smaller relative to the TGF-β pathway. Given the robust effect on XIST levels among all members of the TGF-β superfamily, we focused our attention on this pathway.…”

“…Such XIST modulators [e.g., protein kinase PDPK1 and activin receptor A, type I (ACVR1)] have recently been found among factors that are required for XCI by Bhatnagar et al (22). When we measured XIST levels in Xi-8 cells expressing shRNAs from our screen (Dataset S2), we found that the knockdown of BMP/TGF-β superfamily members induces a robust and consistent downregulation of XIST (Fig.…”

“…Four of the 30 genes identified in our screen, Acvr1, Aurka, Dnmt1, and Pdpk1, were previously identified in a screen using a CMV-driven GFP reporter on the Xi (22). Moreover, these four genes were targeted with the same hairpins in the two screens, as both screens used the same shRNA library.…”

“…Second, the largest functional group we identified consisted of eight genes involved in chromatin structure and regulation, including Dnmt1 and HDAC3, which had previously been linked to XCI (19,32). Third, our screen identified 4 of the 13 genes found by Bhatnagar et al (22) in a screen that used a fundamentally different reporter to identify Xi reactivators, namely GFP driven by the CMV promoter. Finally, our functional screen identified factors involved in sister chromatid cohesion (SCC), whose components were independently identified in a biochemical screen as XIST-interacting factors (20).…”